Dendritic cells (hereinafter briefly referred to as “DC”) are antigen-presenting cells which influence the immune system by interacting with lymphocytes. Most DCs exhibit an immunostimulatory activity. These classical DCs can induce the formation of helper and killer T cells in vivo in different ways (“nature's adjuvant”). In particular, immature DCs which occur in peripheral tissues have the capability of binding antigens and preparing immunogenic MHC peptide complexes therefrom (“antigen processing mode”). Upon the action of maturation-inducing stimulants, such as inflammatory cytokines, these immature DCs develop into potent T-cell stimulants through an increased formation of adhesion and costimulatory molecules (“T-cell stimulatory mode”). At the same time, the cells migrate into secondary lymphatic organs to select and stimulate rare antigen-specific T cells. It could be shown that DCs which were isolated from tissues or blood and loaded with antigen in vitro were immunogenic after back injection as mature DCs in vivo.
Recently, it could be shown that DCs can induce CD4+ and CD8+ T-cell immunity in both healthy humans and cancer patients. In immunocompetent healthy subjects, a single booster injection with mature DCs could enhance not only the frequency, but also the functional avidity of the CD8+ T-cell response. For these reasons, DCs (especially mature ones) are currently extremely promising adjuvants for induce potent T-cell responses against tumors and infections in humans.
One precondition for the use of DCs in immunotherapy is the development of techniques which allow to produce a great number of DCs in culture, either from proliferating CD34+ precursor cells or from non-proliferating or little proliferating CD14+ monocytic precursor cells. DCs derived from monocytes are frequently used currently because they are easily prepared without any cytokine pretreatment of the donor, and because the resulting DC population is fairly homogeneous and best characterized. For example, immature DCs can be prepared from adherent monocytes in the absence of fetal calf serum (FCS) during a culture for usually six to seven days in (GM-CSF+IL-4), followed by maturation for mostly one to three days, induced by autologous monocyte-conditioned medium. To provide an effective cryoconservation for dendritic cells or their precursor cells has proven extremely difficult, except in the presence of FCS (Taylor et al. (1990) Cryobiology 27, 269; Makino et al. (1997) Scand. J. Immunol. 45, 618). However, since FCS must not be present in vaccinations, it has still been necessary to prepare DCs freshly for each DC vaccination, either from fresh blood, from fresh leucapheresis products or from frozen PBMC (“peripheral blood mononuclear cells”) aliquots from leucapheresis products (Thurner et al. (1999) J. Immunol. Methods 223, 1). Frozen PBMCs also have the disadvantage that following the thawing the cells must first be cultured for several days to differentiate them into DCs. Lewalle et al. (2000) J. Immunol. Methods 240, 69-78, disclose a method for the freezing of immature DCs, but only obtain poor yields of surviving cells (p. 71, top of left column). The freezing of immature dendritic cells prepared from monocytes by means of GM-CSF and IL-4 has also been disclosed in WO 99/46984.